Calcium-regulated ion channels control essential processes in nerves and muscles. Channelopathies associated with the voltage-dependent sodium channel family (denoted NaV1.n, VDSC or VGSC) include several severe forms of epilepsy, cardiac Long QT syndrome 3 and ventricular fibrillation, familial autism, pain insensitivity, and other defects in the generation and propagation of action potentials. Most members of the NaV family of channels respond to calcium via regulation by calmodulin (CaM), an essential eukaryotic calcium sensor that is comprised of two highly homologous domains (N and C) connected by a regulatory linker. New solution structures show how apo (calcium-free) C binds to an IQ motif (IQxxx[R,K]Gxxx[R,K]) in the intracellular C-terminal tail of the pore-forming alpha subunit of NaV1.2 (2KXW) and NaV1.5 (2L53). For both, Ca2+ binding to CaM changes the conformation of C and lowers its affinity for the NaV IQ motif. Although these structures and thermodynamic studies show that N does not bind to the NaV IQ motif, genetic studies indicate that mutations in N affect NaV function. Thus, a significant gap in our understanding of this calcium- mediated switching process is how and where N associates with NaV. We hypothesize that CaM serves as a calcium-triggered channel organizer; the N and C domains of CaM bring together distinct parts of NaV to regulate conformation and conductivity. We propose that apo CaM is tethered by C at the IQ motif. When Ca2+ binds to CaM, N binds to its preferred site in NaV, while C either (a) opens and twists on the IQ motif, with the same orientation, or (b) releases and rebinds with opposite orientation. These orientations would restrict the NaV interfaces available to N in different ways. To determine how CaM translates changes in intracellular [Ca2+] into conformational work, and regulates multiple isoforms of NaV, we will determine the distinct roles of N and C by testing how they recognize naturally occurring variations in isoforms of human NaV and how disease-causing mutations in NaV uniquely affect binding of apo and calcium-saturated N and C. We propose three Aims focused on sodium channels found in the central nervous system (CNS). The first Aim will determine how apo and Ca2+-saturated CaM C-domain binds disease-causing mutant IQ motifs of NaV. The second Aim will determine the effect of CaM on interactions between NaV IQ motifs and NaV EF-Hands. The third Aim will determine where apo and (Ca2+)2-CaM N-domain are binding to isoforms of NaV. Structures of CaM will be studied by crystallography, NMR, and CD, while dynamics and energetics from titrations will be determined with fluorescent biosensors encoding known and putative CaM-binding domains, NMR, and calcium titrations monitored by steady-state fluorescence. We will discover key macromolecular orientations between CaM and NaV, provide the first structure of an NaV tail fragment containing both the EF-hand and IQ motif, and indicate the CaM-mediated contribution to multiple disease-associated mutations related to epilepsy, or identified as candidates for autism-spectrum disorders.